Cd44 bv650

Immunophenotypic analyses of splenoctyes from animals were assessed by flow cytometry. Antibodies to stain for MDSC were CD11b-APC (Clone M1/70; BD Biosciences), Ly6G-FITC (Clone 1A8; BD Biosciences), Ly6C-PE (Clone AL-21; BD Biosciences); for dendritic cells were CD11c (Clone HL3; BD Biosciences); for B cells B220-APC (Clone RA3–6B2; BD Biosciences), CD3-FITC (Clone 145–1011; BD Biosciences). T regulatory cells with a phenotype of CD4⁺CD25⁺FoxP3⁺ were evaluated using a commercially available kit (eBiosciences, San Diego, CA). For T cell activation markers, cells were stained with antibodies specific for CD4-PE-Cy7 (Clone RM4–5; BD Biosciences), CD8-PE-Cy7 (Clone 53–6.7; BD Biosciences), CD62L-PE (Clone MEL-14; BD Biosciences), and CD44-Bv650 (Clone IM7; Biolegend). To determine Th1 and Th2 phenotypes, cells were stained using fluorochrome conjugated antibodies targeted CXCR3-PE-Cy7 (Clone CXCR13–173; Biolegend), CCR4-PE (Clone 2G12; Biolegend), and CCR6-APC (Clone CK4-L3; BD Biosciences). Cells were incubated on ice for 30 minutes, washed, and fixed in PBS containing 1% formalin for flow cytometric analysis on a LSRII flow cytometer (BD Biosciences).

Cells were cultured and stimulated in 96-well plates at 1 × 10⁵ cells per well. Plates were coated with anti-CD3 antibody (Biolegend) at concentrations of 0, 0.125, 0.25, 0.5, and 1 μg/ml at 4°C overnight and were washed twice with PBS before incubation. Cells were stimulated in the anti-CD3–coated plates with soluble anti-CD28 at 2 μg/ml (Biolegend). On days 1 and 3, cells were stained with dead cell dye as well as antibodies recognizing the lineage and activation markers CD3 BUV737 (BD), CD4 BUV395 (BD), CD8 PerCP-Cy5.5 (Biolegend), CD69 PE (Biolegend), CD44 BV650 (Biolegend), and CD25 APC (Biolegend). For proliferation assays, CD4⁺ or CD8⁺ T cells were isolated and stained using the CellTrace Violet or CFSE Cell Proliferation Kit (Life Technologies). Briefly, purified cells were washed with PBS and incubated with CellTrace dye for 20 minutes at 37°C protected from light. After 20 minutes, complete RMPI medium was added to the cell suspension, and the cells were incubated 5 minutes further before being washed and resuspended in complete RPMI medium. Cells were cultured in 96-well plates at 1 × 10⁵ cells per well and were stimulated using anti-CD3/CD28 dynabeads (Gibco) at a 1:1 ratio with T cells. On days 1 and 3, cells were stained with dead cell dye, and proliferation was measured at the same time.

CD90.1 BB515 (BD, clone OX-7, cat 564607), CD90.2 BUV395 (BD, clone 53-2-1, cat 565257), CD4 BUV737 (BD, clone RM4-5, cat 565246), CD8 PerCP-Cy5.5 (Biolegend, clone 53–6.7, cat 100734), CD44 BV650 (Biolegend, clone IM7, cat 1033049), CD11b APC (Biolegend, clone M1/70, cat 101212), CD11c APC (Biolegend, clone N418, cat 117310), NK1.1 APC (Biolegend, clone PK136, cat 108710), B220 APC (Biolegend, clone RA3-62B, cat 103212).